This invention relates to novel disulfides and thiols of up to about eighteen amino acids.
The predominant small molecule disulfide in the cell is glutathione. The majority of intracellular glutathione is present in its reduced thiol form, which contributes to intracellular reducing conditions. The intermolecular disulfide in glutathione is far less stable than intramolecular protein disulfides and can rapidly convert between thiol and disulfide. Creighton, T. E., Proteins. New York, N.Y. W. H. Freeman and Co., 1993. Other similar disulfides, are also likely to affect the redox equilibrium. Cellular thiols/disulfides are thought to be important in determination of protein structure by enabling the formation of disulfide bonds between Cys residues via thiol-disulfide exchange. Protein-bound free SHxe2x80x94 groups also play key roles in regulating DNA transcription and binding of regulatory proteins to DNA. A variety of metabolic pathways are significantly impacted by redox state, since thiols have been shown to control the activity of numerous enzymes. Gilbert, H. F., Adv. Enzymol. Metab. Relat. Areas 63:69, 1990; Ziegler, D. M., Ann. Rev. Biochem. 54:305, 1985. Regulation of enzyme systems may play a critical role in maintaining cellular homeostasis and preventing oxidative stress. Reactive oxygen intermediates such as peroxides also have regulatory effects. Therefore, there is a need for new disulfide and thiol molecules that can be potentially useful as agents that affect oxidation-reduction equilibrium in a cell.
It is an object of the present invention to provide novel disulfides and thiols that are small molecules. In one aspect, the compound is of the formula (1): Axe2x80x94Bxe2x80x94Cxe2x80x94Sxe2x80x94Sxe2x80x94Dxe2x80x94Exe2x80x94F, wherein: A and F are selected from the group consisting of hydrogen, an amino acid, a dipeptide, a tripeptide, a modified polypeptide up to three amino acids long, and a carbobenzoxy group, B and E are selected from the group consisting of an amino acid, a dipeptide, a tripeptide, and a modified polypeptide comprising up to and including three amino acids, C and D are selected from the group consisting of a modified polypeptide and a polypeptide comprising up to and including three amino acids, and S is the sulfur atom in the modified polypeptide and the polypeptide in C and D.
In another aspect, the compound is of the formula (II): Axe2x80x94Bxe2x80x94Cxe2x80x94S, wherein: A is selected from the group consisting of hydrogen, an amino acid, a dipeptide, a tripeptide, a modified polypeptide up to three amino acids long, and a carbobenzoxy group, B is selected from the group consisting of an amino acid, a dipeptide, a tripeptide, and a modified polypeptide comprising up to and including three amino acids, C is selected from the group consisting of a modified polypeptide and a polypeptide comprising up to and including three amino acids, and S is the sulfur atom in the modified polypeptide and the polypeptide in C.
In a first embodiment, the present invention relates to novel disulfide and thiol compounds of the Formula I:
Axe2x80x94Bxe2x80x94Cxe2x80x94Sxe2x80x94Sxe2x80x94Dxe2x80x94Exe2x80x94F
Wherein:
A and F independently may be hydrogen, an amino acid, a di- or tri-peptide, a modified polypeptide up to 3 amino acids long, a carbobenzoxy or other ring structure known in the art to facilitate lipid solubility and/or provide other beneficial pharmacologic properties. Hydrophobic amino acids, aromatic amino acids including tryptophan, and phenylalanine, are especially preferred.
B and E independently may be an amino acid, a di or tri-peptide, or a modified polypeptide up to 3 amino acids long. In a preferred embodiment, B and/or E are neutral amino acids.
C and D independently may be a modified polypeptide or traditional polypeptides up to 3 amino acids long. In a preferred embodiment, C and/or D are neutral amino acids.
S denotes the sulfur in the amino acids in C and D.
In a preferred embodiment A, B, C, D, E, F are single amino acids or in some cases a dipeptide. Hydrophobic or hydrophilic amino acids, may be used to alter biodistribution and bioactivity.
The identity of A and F and of each of the lettered moieties in the other pairs may be distinct from the other member of the pair and also from any moiety in any of the pairs. Thus, the molecules may be homo- or heterodimers.
Specific examples of half molecules, which may be combined in a fashion to form dimers include but are not limited to:
Key:
alaxe2x80x94alanine (preferably beta-alanine)
cysxe2x80x94cysteine
cyxe2x80x94cysteamine
In the most preferred embodiment, the beta form of alanine is used in the above examples or as A or B or E or F. D and L forms of all amino acids are also within the scope of this invention.
Any ring structure may be substituted for the Cbz group using any of a variety of linkages known to those skilled in the art. Any relatively neutral amino acid may be substituted for ala; these include b-alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, aspergine, glutamine (to include both D and L forms of these compounds). Any other thiol-containing molecule or other molecule capable of dimerizing may be substituted for cysteine. The present invention includes the half molecules diagramed above and modified half molecules which have an attached phosphate group (xe2x80x94PO3H2) or other similar group. It also includes, but is not limited to phosphate (xe2x80x94OPO3H2), phosphorus acid (xe2x80x94PO3H3), hydrogen sulfate (xe2x80x94OSO3H) sulfonic acid (xe2x80x94SO3H), sulfinic acid (xe2x80x94SO2H), sulfenic acid (xe2x80x94SOH), and metallic salts of these species. In a preferred embodiment, the attached phosphate group or similar group may be removed from the half molecule under physiologic conditions.
In the most preferred embodiment, beta-alanine is used in A, B, C, D, E, and/or F. The following compounds are representative of the novel disulfides and thiols of the present invention: 
And corresponding monomer; 
And corresponding monomers; 
And corresponding monomers; 
All of the compounds of the present invention can be synthesized using routine methods known to those skilled in the art without undue experimentation. Indeed many companies now exist that routinely make small molecules such as those of the present invention on a made-to-order basis based on the structure of the compound alone. The Examples set forth below provide protocols for making certain compounds that are representative compounds of the present invention. Simple variations on the protocols set forth in the Examples can be determined to make other compounds of the present invention by persons skilled in the art.
Beta-alanyl cysteamine disulfide (Betathine(trademark), Beta LT(trademark), BT, also beta-alethine) is a dimer composed of two small thiols which has diverse biological activity. Recently xcex2-alethine has been shown to exhibit potent antitumor activity in vivo. For example, in an NS-1 mouse myeloma model, repeated administration of xcex2-alethine as a monotherapy soon after inoculation with relatively low doses of tumor significantly increased survival rates. Treatment with xcex2-alethine was also found to increase the percent survival in the Cloudman S-91-DBA/2 model in which melanomas had already been established. U.S. Pat. No. 5,643,966; U.S. Ser. Nos. 08/468,043; 08/468,041; 08/346,177. xcex2-alethine has been used as an adjunct to chemotherapy in two aggressive murine tumor models, where it was admin along with melphalan in the treatment of the MOPC-315 myeloma and with cyclophosphamide in the treatment of the B16 melanoma, and it was found to be a beneficial adjunct to chemotherapy in both systems. U.S. Provisional Application Nos. 60/075,966 and 60/085,474, which are incorporated herein in their entirety. Despite the growing body of in vivo studies showing the antineosplastic effects of BT, the underlying mechanism had not been determined.
Beta-alethine and certain other thiols and disulfides (described in U.S. Ser. No. 08/733,174) have also been used as adjuvants in vaccines and as immunostimulatory molecules. Further, the compounds beta-alanyl taurine and carbobenzoxy beta-alanyl taurine and related compounds collectively called Taurox(trademark) have been used as anti-cancer agents (U.S. Pat. Nos. 5,370,818; 5,578,313), for the therapeutic treatment of immune diseases (U.S. Ser. Nos. 08/466,143; 08/469,697); and in cell culture and therapy (U.S. Ser. Nos. 08/463,732 and 08/463,784). All of the above patents and applications are incorporated by reference into this application in their entirety.
Without meaning to be bound by theory, the structure of xcex2-alethine suggests that its disulfide moiety may be crucial for activity. The predominant small molecule disulfide in the cell is glutathione. The majority of intracellular glutathione is present in its reduced thiol form, which contributes to intracellular reducing conditions. The intermolecular disulfide in glutathione is far less stable than the typical intramolecular protein disulfides and can rapidly convert between the thiol and disulfide forms. Other similar disulfides, such as BT, and their corresponding thiols, are also likely to affect the redox equilibrium. Cellular thiols/disulfides are thought to play an important role in determining protein structure by enabling the formation of disulfide bonds between Cys residues via a thiol-disulfide exchange. A variety of metabolic pathways are also known to be significantly impacted by redox state, and thiols have been shown to control the activity of numerous enzymes